The overall objective of this project is to determine the structural, biochemical and physiological processes involved in hard tissue formation with particular emphasis on mineralization. The specific aim of the research for the proposed granting period is to investigate the role of highly phosphorylated proteins in the mineralization process. Phosphoproteins are demonstrated intermediates in the mineralization of both vertebrate dentin and invertebrate bivalve shells and postulated intermediates in bone and enamel mineralization. The mineralization model selected for this study is the extrapallial fluid-shell system of bivalve molluscs. Although bone and teeth are of far greater medical import than invertebrate shells, the study of normal and pathological mineralization of both hard and soft tissues in the unamenable vertebrates will be facilitated if the details of the physiological process are understood in at least one relatively simple amenable system. The scientific advantage of working with some species of bivale molluscs lies in the fact that these animals have a continuously developing mineralized exoskeleton (i.e. the shell) in dynamic equilibrium with a relatively large body of fluid (i.e. the extrapallial fluid) which can be tapped for mineral ion carries and the requisite cells, enzymes, and structural proteins involved in the physiological process of mineralization. The role of the highly phosphorylated proteins (which occur as discrete mineral ion-sequestering particles in clams) will be investigated from their intracellular synthesis to their action at the mineralization front. The experimental approach will range from the purely chemical to the very biological. Specifically the methodology includes: 1. Determination of the cell population responsible for the phosphoprotein synthesis. 2. Isolation and characterization of the phosphoprotein particles and monomers from the cells. 3. Determination of the source of the protected mineral pool within the phosphoprotein paricles. 4. Determination of the rate of phosphoprotein incorporation into the developing shell layers during periods of rapid mineralization. 5. Isolation and characterization of modified phosphoprotein particles from the mineralization front. 6. Isolation of the membrane limited vesicles occurring in the extrapallial fluid and determining whether or not they contain enzymes capable of modifying the phosphoprotein particles. 7. Utilization of mineral ions prepackaged as mineral ion-phosphoprotein complexes in shell development. 8. Identification of solid calcium phosphate mineral in recently formed shell layers. 9. Investigation of specifc domains within the phosphoprotein with respect to amino acid sequences and mineral ion affinity.